Shear Dependent LC Purification of an Engineered DNA Nanoswitch and Implications for DNA Origami
Shear Dependent LC Purification of an Engineered DNA Nanoswitch and Implications for DNA Origami
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DOI：
10.1021/acs.analchem.7b00791
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发表时间：
2017-06-06
影响因子：
7.4
通讯作者：
Basanta-Sanchez, Maria
中科院分区：
文献类型：
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作者：
Halvorsen, Ken;Kizer, Megan E.;Basanta-Sanchez, Maria
随着DNA纳米技术的成熟，人们越来越需要快速、可靠和自动化的纯化方法。在这里，我们开发了UHPLC方法来纯化自组装的DNA纳米开关，这些纳米开关是使用DNA折纸方法形成的，并且被设计为响应于结合伴侣而改变构象。我们发现剪切降解阻碍了DNA纳米开关的LC纯化，从支架链上去除寡核苷酸并导致功能丧失。然而，柱、流速和缓冲液的适当选择使得能够在半小时内稳健地和自动地纯化DNA纳米开关而不丧失功能。将我们的方法应用于DNA折纸结构，我们发现类似于400 nm长的纳米管在最温和的流动条件下降解，而类似于40 nm直径的纳米球即使在侵蚀性条件下也保持完整。这些例子显示了在LC纯化过程中流体应力如何影响不同的DNA纳米结构，并表明剪切力可能与DNA纳米技术的某些应用有关。这种方法的进一步发展可能会导致各种形状和大小的DNA纳米结构的快速和自动化纯化，这将是该领域的重要进展。
As DNA nanotechnology matures, there is increasing need for fast, reliable, and automated purification methods. Here we develop UHPLC methods to purify self-assembled DNA nanoswitches, which are formed using DNA origami approaches and are designed to change conformations in response to a binding partner. We found that shear degradation hindered LC purification of the DNA nanoswitches, removing oligonucleotides from the scaffold strand and causing loss of function. However, proper choice of column, flow ratel, and buffers enabled robust and automated purification of DNA nanoswitches without loss of function in under a half hour. Applying our approach to DNA origami structures, we found that similar to 400 nm long nanotubes degraded under the gentlest flow conditions while similar to 40 nm diameter nanospheres remained intact even under aggressive conditions. These examples show how fluid stresses can affect different DNA nanostructures during LC purification and suggest that shear forces may be relevant for some applications of DNA nanotechnology. Further development of this approach could lead to fast and automated purification of DNA nanostructures of various shapes and sizes, which would be an important advance for the field.